Cell-matrix mechanical interactions drive fundamental processes such as developmental morphogenesis, wound healing, and remodeling of bioengineered tissues. The overall goal of this research is to determine the underlying biochemical and biophysical mechanisms which regulate these critical processes in corneal fibroblasts. In the first funding period, we developed a new experimental model for directly investigating cell-matrix mechanical interactions inside 3-D fibrillar collagen matrices. Data obtained using this innovative approach has provided new insights into potential mechanisms of sub-cellular force generation, matrix remodeling, and the modulation of cell behavior by mechanical stress which together lead to the following Hypotheses: 1) Rac induces spreading of corneal fibroblasts via localized tractional force generation by extending pseudopodia, whereas Rho induces contractile force generati0n along the cell body; these effects [unreadable] are mediated by differences in sub-cellular regulation of myosin light chain phosphorylation, 2) corneal [unreadable] fibroblasts actively respond to increases or decreases in local matrix stress in order to maintain tensional homeostasis (constant tension); these responses are mediated by compensatory and reciprocal changes in Rho and Rac; and 3) the pattern and amount of permanent collagen matrix remodeling is maximal in stationary, contractile cells (high Rho and low Rac activity), and is enhanced by cell-cell mechanical communication at higher densities. To test these hypotheses, we propose the following Specific Aims: 1) determine the role of Rho and Rac in regulating cytoskeletal organization, mechanical behavior, and sub-cellular force generation by corneal fibroblasts inside 3-D matrices using our time-lapse imaging system, 2) investigate the mechanical response of corneal fibroblasts to changes in ECM stress by inserting microneedles into the ECM next to isolated cells, in order to modulate matrix stiffness; and, 3) compare the pattern and amount of local collagen matrix remodeling by corneal fibroblasts at different cell densities and culture conditions. Accomplishing these Aims should provide a better understanding of how the fundamental processes of cell spreading, contraction and matrix remodeling are regulated. This is an important step towards our long-term objective of controlling the organization and mechanical behavior of corneal fibroblasts inside artificial matrices [unreadable] through the design of "smart" 3-D ECM scaffolds that incorporate both biochemical and mechanical cues. [unreadable] [unreadable]